The long-term objectives of this proposal are to generate and commercialize a new class of high-specificity, high-affinity proteins called Lectenz, as research reagents for use in studies of disease- or cancer-related glycosylation. The specific aims are to further the development and commercialization of a new reagent, created in Phase I of this proposal, which is pan-specific for the detection of the glycan &#946;-O-GlcNAc. By basing the design of this reagent on the biologically-relevant O-GlcNAcase enzyme, the resultant Lectenz should be able to recognize the terminal O-GlcNAc glycan in its biologically-relevant context. For this reason, we refer to this Lectenz as being pan-specific for O-GlcNAc. In this Phase II proposal, we will fully characterize this unique reagent and optimize its production and utility in a variety of assay formats. The ability of the pan-specific O-GlcNAc Lectenz to rapidly confirm the presence of &#946;-O-GlcNAc in proteins and tissues, without the need to turn to more-elaborate techniques, would provide a powerful tool to delineate differentially glycosylated proteins, and eventually establish correlations between &#946;-O-GlcNAc regulation and associated disease states, such as cancer. In addition, as reported in Phase I, we have found that some clones of this reagent display variations in affinity for O-GlcNAcylated peptides, as a function of the peptide sequence. Thus, we will also continue to select for variants of the Lectenz that may be used to discriminate between &#946;-O-GlcNAcylated peptides, as a function of the peptide sequence.